Nucleic acid sequence amplification technology has a wide application in bioscience, genetic engineering, and medical science for research and development and diagnostic purposes. In particular, the nucleic acid sequence amplification technology using PCR (hereafter referred to as “PCR amplification technology”) has been most widely utilized. Details of the PCR amplification technology have been disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188.
Various apparatuses and methods incorporating automated PCR amplification processes have been developed and used for fast and efficient amplification of a variety of genetic samples. The basic working principle of such technology is as follows.
In the commercialized PCR amplification technology, a sample is prepared to contain a template DNA to be amplified, a pair of oligonucleotide primers complementary to a specific sequence of each single strand of the template DNA, a thermostable DNA polymerase, and deoxynucleotide triphosphates (dNTP). A specific portion of the nucleic acid sequence of the template DNA is then amplified by repeating a temperature cycle that sequentially changes the temperature of the sample. Typically, the temperature cycle consists of three or two temperature steps, and the amplification processes during the temperature cycle occur in the following manner.
The first step is the denaturation step in which the sample is heated to a high temperature and double stranded DNAs become separated to single stranded DNAs. The second step is the annealing step in which the sample is cooled to a low temperature and the single stranded DNAs formed in the first step bind to the primers, forming partially double stranded DNA-primer complexes. The last step is the polymerization step in which the sample is maintained at a suitable temperature and the primers in the DNA-primer complexes are extended by the action of the DNA polymerase, generating new single stranded DNAs that are complementary to each of the template DNA strands. The target nucleic acid sequences as selected by the sequences of the two primers are replicated during each cycle consisting of the above three steps. Typically, several millions or higher number of copies of the target nucleic acid sequences can be produced by repeating the temperature cycles for about 20 to 40 times.
The temperature of the denaturation step is typically 90-94° C. The temperature of the annealing step is controlled appropriately according to the melting temperatures (Tm) of the primers used, and it typically ranges from 35 to 65° C. It is typical to set the temperature of the polymerization step to 72° C. and use a three-step temperature cycle, since the most frequently used Taq DNA polymerase (a thermostable DNA polymerase extracted from Thermus aquaticus) has the optimal activity at that temperature. A two-step temperature cycle in which the polymerization temperature is set to the same as the annealing temperature, can also be used since the Taq DNA polymerase has a broad temperature range of the polymerase activity.
In the most widely used method, a reaction vessel containing the sample is made in contact with a solid metal block having a high thermal conductivity, and the temperature of the solid metal block is changed by combining it with heating and cooling devices to achieve the desired temperature cycling of the sample. The commercial products adopting this type of methods often use a gold-plated silver block that has very high thermal conductivity and/or the Peltier cooling method in order to achieve rapid temperature change. Recently, methods using a fluid such as gas or liquid as a heat source instead of the solid metal block, have been developed to achieve rapid temperature change, and products using such methods are being commercialized. In this type of methods, a fluid heated to a suitable temperature is circulated around the reaction vessel in a manner that an efficient thermal contact can be provided between the fluid heat source and the reaction vessel containing the sample. Other types of methods have also been developed to achieve rapid temperature cycling. Additional examples include a method of contacting the reaction vessel containing the sample or the sample itself sequentially with multiple heat sources each at a specific temperature, a method of heating the sample directly with infrared radiation, etc.
The prior nucleic acid sequence amplification apparatuses have a number of drawbacks as they operate to change the temperature of the whole sample according to the three- or two-step temperature cycle.
Firstly, the prior nucleic acid sequence amplification apparatuses of the temperature cycling type are complex in their design since processes for changing the sample temperature are necessary. In order to perform such temperature change processes, the method incorporating a solid metal block or a fluid as a heat source requires a means for controlling and changing the temperature of the heat source rapidly and uniformly and also a means for controlling the time interval of the temperature change. Similarly, the method of contacting the reaction vessel or the sample sequentially with multiple heat sources each at a specific temperature requires a means for moving the reaction vessel or the sample quickly and precisely and also a means for controlling the moving time and interval.
Secondly, it is difficult to integrate the prior nucleic acid sequence amplification apparatuses in a complex apparatus or a miniaturized device, due to their complicated design. Recently, miniaturized complex apparatuses are under development in the biotechnology field. For example, Lab-on-a-chip has been developed by integrating channels for sample passage, valves, pressure gauges, reaction vessels, detection units, etc. as a single unit on a glass, silicon, or polymer plate using photolithography. Such miniaturized complex apparatuses are expected to have wide applications for various research and medical purposes. In the case that a nucleic acid sequence amplification apparatus needs to be integrated to such miniaturized chip, the prior method has a drawback in miniaturization because it requires a complex design to enable the temperature change processes. Furthermore, it is difficult to integrate the prior apparatuses in a complex apparatus in which rapid temperature change is not desirable.
Thirdly, the prior nucleic acid sequence amplification apparatuses can only use thermostable DNA polymerases such as Taq DNA polymerase. This is because the prior apparatuses have the process of heating the whole sample to a high temperature.
Finally, the prior nucleic acid sequence amplification apparatuses have a limitation for reducing the PCR reaction time. Since the prior apparatuses require the processes for changing the temperature of the whole sample, the PCR reaction time must take more time at least as much as the time needed for the temperature change.